فصلنامه بیماریهای گیاهی
سال پنجاه و یکم شماره 4 (زمستان 1394)

In recent years brown blotch of cultivated mushroom (Agaricus bisporus) has been observed as a prevalent disease in Iran. The disease symptoms of the cultivated agaric include brown blotch, brown pit spots, rotting, weeping and drippy gill. Pseudomonas tolaasii has been cited as the causal agent of bacterial blotch, but much controversy exists regarding the involvement of this bacterium alone or blotch may be caused by more than one organism. In 2011, the diseased samples of mushroom were collected from Khorasan Razavi, Alborz, Tehran, Semnan, Ghazvin and Golestan provinces. Strains which were pathogenic on the cap of mushroom (pileus) were selected for further analysis. Biochemical, physiological and nutritional tests together with amplification of 1Kb region of 16S rDNA using Ps forward and reverse primers showed that all strains belong to Pseudomonas tolaasii. The isolated strains displayed different degrees of pathogenicity on mushroom. The presence of different virulence factors were evaluated among the strains. The results indicated that those strains which showed enhanced pyoverdin production had higher pathogenicity on agaric caps. There was no correlation between the existence of other virulence factors such as biofilm formation, motility, or PQS production and pathogenicity. Our results indicated that in P. tolaasii similar to other Pseudomonads, PQS exists but the actual role of this signal is not so clear. PQS dependent quorum sensing in P. tolaasii could not affect pathogenicity of this bacterium, thus most of the virulence determinants in this bacterium should be PQS independent.

In this study, the bio-control activity of Trichoderma harzianum i25 on root-knot nematode Meloidogyne javanica in the laboratory and greenhouse conditions and its ability to induce biochemical defense enzymes such as peroxidase (POX) and polyphenol oxidase (PPO) and total phenol on tomato were investigated. At first, the nematode by the help of morphological and molecular characters and then fungal isolate using morphological one were identified. Infection ability of fungal isolate on the eggs of M.javanica and effects of fungal culture filtrate on egg hatching and mortality of M. javanica juveniles (J2) were tested in vitro experiment. In the greenhouse experiment, effect of the fungus on tomato plants growth parameters as well as nematode population examined. Also in another experiment, host plant roots at 6 leaf stage were inoculated with fungal spore suspension and nematode as well and POX, PPO and total phenol activity measured after 1 to 8 days of inoculation. Results indicated that the fungal isolate prohibited larval egg hatching and increased nematode mortality and also parasitized the eggs in the egg masses. In the greenhouse, the fungal isolate reduced disease and suppressed nematode population at 85% and also the fungus increased host plant growth parameters. The amount of POX and PPO increased and this increase was at its max. in the 4th and 5th day of the infected plant treated with the fungus. Inoculation of the tomato seedlings with the fungus increased the total phenol amount in the leaves in which the highest amount recorded in the infected fungus treated plant on the 8th day. Results indicated the potential of the isolate i25 in nematode bio-control.

The application of recombinant DNA leads to production of a large number of therapeutic proteins and peptides. Lactoferricin, a strong cationic peptide N-terminal region of lactoferrin, is produced by a protein-digesting enzyme pepsin. So, this project was done to produce lactoferricin of camel in hairy roots of tobacco and investigate its antibacterial effect. For this reason camel lactoferricin peptide gene sequence was extracted from gene bank and after optimization of codon usage synthesized artificially. The mentioned sequence was subcloned into pBI121expression vector. The recombinant pBI121 vector was transferred to Agrobacterium rhizogenes bacteriumand then was used to produce hairy roots. Hairy roots was evaluated and proved by specific primers of gene which coded lactoferricin peptide. Recombinant peptide of lactoferricin was extracted from hairy roots and evaluated against plant pathogenic bacteria including Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas syringae pv. syringae, Erwinia amylovora, Xanthomonas citri, Pectobacterium carotovorum by disk diffusion method. The results showed that this peptide was expressed in hairy roots and has significant antibacterial effect.

Four nematode species of the infraorder Tylenchomorpha namely Paratylenchus microdorus, Filenchus hamuliger, Psilenchus aestuarius and Tylenchus arcuatus collected from the rhizosphere of different plants in Khouzestan province, southwestern Iran, andcharacterized morphologically and morphometrically. The recovered population of P. microdorus is characterized by its 268-386 µm long body, 13-17 µm long stylet and rounded tail terminus. Iranian population of the F. hamuliger is characterized by having two lines in the lateral field, truncated conical head, 8-10 µm long stylet, axial spermatheca and hook-shaped tail terminus. The population of P. aestuarius is characterized by lacking of annuls on head region, having of post anal sac and clavate tail, and finally, the studied population of T. arcuatus is characterized by its 652-809 µm long body, continuous annulated head, elongate conical hooked tail with sharp or slightly rounded terminus. The three species F. hamuliger, P. microdorus and T. arcuatus are new records for the Iranian nematofauna.

Ascospore germination was examined in Polystigma amygdalinum, incitant of red leaf blotch of almond with the goal of more detailed study of the biology and disease phases of this biotroph. Appressorium development is described for a species of Polystigma for the first time. Temperature had marked effect on ascospore germination and appressorium formation process. Germination and appressorium formation occurred in a temperature range of 5 to 20°C. Germination percentages were highest at 5 to 10°C. Ascospores at 25°C showed abnormal swellings and distortion of the cell content. Light had no effect on ascospore germination. The highest germination of ascospores was obtained on potato dextrose agar medium.

In order to investigate the Pythium flora in cereal fields of Fars Province of Iran, During 2012-2013 soilfrom cereal (rice, maize, barley and millet) fields in various parts of Fars Province (Arsanjan, Bajgah, Eqlid, Jahrom, Kamfiruz, Kavar, Kazerun, Khorambid, Larestan, Mamsani Meymand, Naghsh-e-Rostam, Sepidan, Surmaq, Seydan, Sivand and Zarghan) were sampled. Thirteen Pythium species were identified based on morphological, physiological and phylogenetic studies, including: Pythium adhaerens, P. amasculinum, P. aphanidermatum, P. carolinianum, P. coloratum, P. dissotocum, P. diclinum, P. kashmirense, P. marsipium, P. nunn, P. oligandrum, P. periplocum and Pythium sp. “hordeum”. P. adhaerens, P. carolinianum, P. dissotocum, P. kashmirense, P. marsipium, P. nunn and Pythium sp. “hordeum”werenewto Iran flora. Pythium sp. “hordeum”wasconsidered as a new species.

Beet severe curly top virus (BSCTV, recently named BCTV-Svr) and Beet curly top Iran virus (BCTIV) are the causal agents of curly top disease in sugar beet and several other dicotyledonous plants in Iran. In this investigation, genetic diversity and prevalence of curly top viruses in relation to host, geographical distribution and age of the main host plant (sugar beet) were studied. Polymerase chain reaction using specific primer pairs to amplify a part of the genome of BSCTV and BCTIV was applied to 734 plant samples collected from various hosts in five provinces in Iran. DNA fragments approximately 500 and 700 bp in size were amplified from 18 BSCTV and 10 BCTIV infected plants, respectively. Amplified fragments were subjected to sequencing and phylogenetic analysis. Effect of geographical location on genetic diversity of viruses was observed only for BCTIV isolates. Type of host plant had no correlation with genetic diversity of either BSCTV or BCTIV. The highest incidence of both viruses occurred in Fars province. Nonparametric analysis of PCR data showed that the prevalence of each virus varied in different fields and depended on the host, date of cultivation and geographical region. While the incidence of BCTIV in sugar beet plants was higher than BSCTV, BSCTV was more frequent in other plants such as tomato, pepper and bean.

Beet curly top Iran virus (BCTIV, genus Becurtovirus) and Beet severe curly top virus (BSCTV-IR, genus Curtovirus) induce beet curly top disease in different regions of Iran. In addition to sugar beet, tomato and pepper are important hosts of these viruses. Also tomato yellow leaf curl disease (TYLCD) is a serious disease of tomato in Iran. The Iranian isolate of Tomato yellow leaf curl virus (TYLCV-[Ab]), a severe strain of TYLCV (genus Begomovirus), is a major component of TYLCD in southern Iran. Until now, no information is available regarding comparative host range of these viruses. To determine the natural hosts of BSCTV-IR, BCTIV and TYLCV-[Ab], samples of sugar beet, tomato, pepper, turnip and spinach were collected from different regions in Fars province. For the experimental host range determination, a number of plants were grown in the greenhouse and their seedlings were agroinoculated with the infectious clone of each virus. The results showed that BSCTV-IR has a wider natural and experimental host range compared to BCTIV. These results indicated that member of Solanaceae, Brassicaceae, Fabaceae and Amaranthaceae are most frequently infected by BSCTV-IR and BCTIV. Natural host range of TYLCV-[Ab] seemed to be very narrow as, in the present study, this virus was isolated only from tomato. However, under greenhouse conditions, bean, nasturtium, petunia, redroot pigweed, datura, night shade and ground cherry were found to be infected by this virus.

Rice bacterial brown stripe, caused by Acidovorax avenae subsp. avenae is recognized by producing water-soaked and brown stripes on leaves and sheaths of rice seedlings in nursery. Contamination of ecosystems upon excessive use of pesticides and emergence of resistance in pathogens to these chemicals makes continuous research on development of new control methods and strategies to combat plant pathogens an essential task. Plant disease resistance genes are useful genetic resources that can be employed to develop resistant varieties as the best alternative to other control measures. This research aimed to study the role of NH1, PR1, POX and PR10, pathogenesis related proteins and PDR20 gene in two Iranian rice cultivars inoculated with an incompatible strain of bacterial brown stripe using the Quantitative Real-time PCR technique. After Screening of 5 Iranian rice cultivars, Tarom and Sahel were selected as susceptible and resistant cultivars, respectively. The results of this study showed that the expression level of thesegenes has greatly increased in Sahel cultivar in comparison to Tarom (susceptible) cultivar. Increased expression level of the aforementioned genes, proves the role of these genes in resistance of rice plants against bacterial brown stripe disease.

Phytophthora drechsleri, P. cryptogeaand P. erythroseptica are phylogenetically closely related Oomyceteous plant pathogens which are morphologically similar. In order to discriminate these taxa from each other and from species with convergent morphological characteristics a simple as well as a nested-PCR based method was developed. A collection of isolates of each species from different hosts representing world-wide diversity of species were examined for unique regions of nuclear as well as mitochondrial genes. Six candidate PCR primers were designed and calibrated for species-specific amplification of P. drechsleri based on the DNA sequences of rDNA internal transcribed spacer regions and the cytochrome c oxidase subunit I, and also three candidate PCR primers specific for P. cryptogeaand P. erythroseptica were designed and calibrated based on cytochrome c oxidase subunit I. Studies showed that the best primer set for identification of P. drechsleri was the combination of ITS-DF2 and ITS-DR2, which amplified a 567 bp band. The combination of COX-CF1 and COX-CR2 was the best set for discrimination of P. cryptogea/P. erythroseptica from other species which amplified a 415 bp product from both species. A restriction map analysis of P. cryptogea/P. erythroseptica indicated that the non-palindromic Mnl I enzyme restriction site was unique to amplicons of P. erythroseptica isolates and could be employed to distinguish this species from P. cryptogea. Based on this study, nested-PCR was at least 100 times more sensitive than conventional PCR for detection of these species.

During May 2011 to September 2012 soil samples from Shiraz landscape were collected,air dried and passed through a 2-mm sieve and baited with sugarbeet seeds. Twenty seven isolates of Rhizoctonia were isolated from soil, which 20 isolates belonged to Rhizoctonia solani AG2 (6 isolates), AG4 (7 isolates), AG2.2, AG7 and AG13 (each one isolate) and 7 isolates of binucleate Rhizoctonia (BNR). Also eleven isolates ofR. solani were isolated from organic matter contained of AG2 (3 isolates), AG4 (4), and AG7 (1). Anastomosis group of some isolates of R. solani and all isolates of BNR due to unavailability of tester isolates could not be identified.

Huanglongbing (HLB) also known as citrus greening is one of the most destructive disease of citrus worldwide. Three bacterial species including ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’ and ‘Ca. L. americanus’ have been identified as causal agents associated with this disease (Bové 2006). ‘Ca. L. asiaticus’, agent of Asiatic form of the HLB disease has been previously reported in Sistan-Baluchistan, Hormozgan (Faghihi et al. 2008) and Kerman (Mohkami et al. 2012) provinces. Based on the presence of Asian citrus psyllid, vector of ‘Ca. L. asiaticus’ and exhibition of Huanglongbing-like symptoms including mottling of leaves and yellowing of shoots in citrus growing areas of Fars province, samples of psyllids and symptomatic citrus trees were tested for the presence of HLB using PCR assay. Direct PCR using F1/R1 primer pair and nested PCR using primer pairs F1/R1(first round) and F2/R2 (second round), specific for Asiatic form of HLB, resulted in amplification of expected fragments of 535 and 400 bp, respectively from six out of 14 psyllid and three out of 10 Lisbon lemon samples in Renjaco (Mohammadabad, Jahrom), 10 out of 14 psyllid samples in Dashte-peergheyb (Darab), five of eight Valencia sweet orange and three of five grapefruit samples in Tange Eij (Fasa) (Fig. 1). No amplicons were obtained from symptomless sweet orange trees and psyllid samples reared on healthy sweet orange seedlings. Direct PCR-amplified fragment from a psyllid and a Lisbon lemon sample from Jahrom were directly sequenced and submitted In GenBank database under accession numbers KT626605 and KT626604 respectively, BLAST search using obtained sequences showed 99% identity with corresponding sequences of ‘Ca. L. asiaticus’ rplKAJL-rpoBC operon from Hormozgan province (GenBank accession number, FJ172759) and other areas in the world (GenBank accession numbers, M9439, AY34200 and EU078703). This is the first report on the occurrence of HLB disease in Fars province. HLB in Fars province is a serious threattocitrus productioninadjacent provinces